Uzarigenin glycosides



United States Patent 3,148,182 UZARIGENKN GLYCOSIDES Andor Fiirst, Basel, Werner Meier, Binningen, and Kuno Meyer, Basel, Switzerland, Camila A. R. Gavilanes, Las Palrnas, Canarias, Spain, and Richard Rees, Los Angeles, Calif, assignors to Hofimanu-La Roche Hue, Nutley, N.J., a corporation of New Jersey No Draw ng. Filed Mar. 1, 1961, Ser. No. 92,469 Claims priority, application Switzerland Mar. 4, 1960 4 Claims. (Cl. 260-2105) The present invention relates to processes for obtaining cardioactive glycosides from Digitalis canariensis L. var. isabelliana (Webb) Lindinger, hereinafter designated Digitalis isabelliana. More particularly, it relates to processes for obtaining crude extracts contaning the main glycosides of this plant, and for isolating from said crude extracts two hitherto unknown uzarigenin glycosides.

The processes according to the invention comprise extracting leaves of Digitalis isabelliana with water and aqueous alcohol, precipitating ballast materials from the aqueous alcoholic solution, extracting the thus purified aqueous alcoholic solution in turn with ether, chloroform, and chloroform/ alcohol mixture consisting of one part by volume of alcohol and four parts by volume of chloroform, isolating the active materials which have passed into the chloroform/alcohol mixture, and (if desired) isolating from the thus obtained crude extract, which contains uzarigenin glycosides and digitoxigenin glycosides, the two novel uzarigenin glycosides in the form of mixed crystals thereof.

Digitalis isabelliamz (synonym: Callianassa isabelliana) employed as the starting material is a kind of digitalis, of exceptionally high activity, indigenous to Grand Canary Island. Thus, upon determination of toxicity according to Hatcher-Magnus, it has been found that a cold extract of the leaves of Digitalis isabelliana is con siderably more toxic than corresponding extracts from Digitalis pm'purea and Digitalis larzata.

It has been found that the unusually high activity of Digitalis isabelliana is due on the one hand to a glycoside content higher than the average, and on the other hand to the presence of hitherto unknown cardioactive glycosides. The highest activity of the various extracts obtained by the processes of the invention is exhibited by a 4:1 chloroform/alcohol extract, see above. In said extract are found the main glycosides of the plant. Upon removal of the solvents, the active material is obtained in the form of an amorphous, greenish-brown powder. The crude extract contains, as can be demonstrated by paper chromatography, at least five components having a positive Kedde reaction (violet-blue color reaction with dinitrobenzoic acid and alkali, characteristic for compounds of the cardenolide type).

It has been further found that two glycosides predominate in the crude extract. They are designated herein as Glycoside A and Glycoside B. These two glycosides can be obtained from the crude extract in the form of water-soluble mixed crystals having MP. of about 270-276". It has been found that Glycosides A and B contain uzarigenin as the aglycone.

The lethal dose of the above mentioned mixed crystals (as determined according to Hatcher-Magnus in cats by intravenous administration) is 0.45:0.05 mg./kg., i.e. considerably below the lethal dose of uzarigenin glycosides mentioned in the literature, e.g. odoriside B, uzarin or cheiroside A.

By the action of the glucose-splitting enzyme strophanthobiase, glucose can be split oil? from Glycosides A and B (obtained as mixed crystals), whereupon there are obtained two uzarigenin monosides, hereinafter designated as Desgluco-glycoside A and Desgluco-glycoside B; the

3,148,182 Patented Sept. 8, 1964 latter can be separated from each other and obtained in crystalline form.

Desgluco-glycoside A Desgluco-glycoside B -17.2;(=2 (c=1.09 in Methanol).

0.74 (in system chloroform/formarnide) Rr-Value form/formamlde) car Glucose-digitoxose-O 4 The processes of obtaining crude extracts and mixed crystals, taught by the present invention, as well as the degradation reactions taught herein, can be illustrated schematically as follows:

Digitalis isabelliana Crude Extract (from chloroform/alcohol 4:1)

Mixed Crystals Glycoside A (fermentative splitting-01f of g ucose) Desgiuco-glycoside A DesgZuco-glycoside B (nzarigenin-digitoxoside) (Splitting-01f of the 2-desoxy sugar which is combined with the aglycone) Uzarigeni'n In the mother liquors remaining after isolation of the mixed crystals from the crude extract, there can be found, in addition to residual amounts of the two Glycosides A and B, additional digitoxigenin glycosides. Thus, by the action of strophanthobiase, the following additional monosides are found:

M.P. Evatrornonoside 210-213 Digitoxigenin gluomethyloside 225-230 Digitoxigenin allomethyloside 152-156 Digitoxigenin glucomethyloside 222-224 The crude extracts obtained according to the processes of the invention, the mixed crystals isolated therefrom, and the constituent glycosides thereof, are usful as medicinal agents, more particularly as cardioactive and diuretic agents.

The invention is further disclosed in the following ex- Glycosiie B ample, which is illustrative but no lirnitative thereof. Temperatures are in degrees centigrade.

Example A mixture of 500 g. of pulverized leaves of Digitalis isabelliana and 800 ml. of Water is heated to 60 and stirred at this temperature for 90 minutes. Then 800 ml. of ethanol is added, the mixture is stirred for an additional period of 90 minutes at 60, and filtered. The residue is extracted first with 1000 ml. of 60% ethanol, then with 1000 ml. of 70% ethanol, and finally is extracted twice, each time with 1000 ml. of 90% ethanol. The various extracts are combined and concentrated in vacuo to a volume of 700 ml. The concentrate obtained is diluted with 700 ml. of ethanol, and the ballast materials are precipitated by addition of lead (II) hydroxide. After separating the precipitate, the aqueous alcoholic solution is treated with the quantity of hydrogen sulfide required to precipitate the lead, and the lead sulfide is filtered oil". The thus purified extract is concentrated in vacuo to 500 ml. and extracted in turn with ether (total of 1.5 liters), chloroform (total of 2.5 liters), and a mixture of four parts by volume of chloroform and one part by volume of ethanol (total of 2.5 liters). The aqueous phase is then concentrated to 400 ml. and extracted with a mixture of two parts by volume of chloroform and one part by volume of ethanol (total of 4 liters).

In the following table are shown the quantities of substances obtained from the various extracts, as well as their i.e., more than 112 mgjkg.

The crude extract (210.2 g.) isolated from the chloroform/alcohol mixture (4:1) is an amorphous greenishbrown powder, containing uzarigenin glycosides and digitoxigenin glycosides. As shown in the table, this crude extract exhibits higher activity than all the other fractions. Upon paper-chromatographic development (system chloroform/tetrahydrofuran/formamide 50:50:65 parts by volume, descending, Whatman No. 1 filter paper impregnated with formamide), it is found that at least five compounds having positive Kedde reaction are contained in the crude extract, the two uzarigenin glycosides A and B being predominant in quantity.

The two glycosides can be obtained in the form of mixed crystals from the above described crude extract as follows:

20.2 g. of crude extract is dissolved in 150 ml. of

acetone, the solution is concentrated in vacuo until cloudiness appears, and then is cooled in an ice bath for three hours. 4.3 g of a gray-green powder separates, having M.P. 282-284". Upon recrystallization from aqueous methanol, there are obtained fine colorless needles having M.P. 270-276". This product is a crystalline mixture of Glycosides A and B. -It is water-soluble. The lethal dose determined according to Hatcher-Magnus is 0.45 i 0.05 rug/kg. i.v.

By means of strophanthobiase, glucose can be split off from mixed crystals of Glycosides A and B isolated in the form of mixed crystals, whereupon there are obtained two monosides (Desgluco-glycoside A and Desglucoglycoside B).

Upon hydrolysis with 0.05 N sulfuric acid in 50% methanol, these two monosides can be split up into the aglycone uzarigenin and to the Z-desoxy sugar linked directly to the aglycone. In the case of Desgluoo-glycoside B, D(+)digitoxose is thereby obtained.

We claim:

1. Water-soluble mixed crystals of two uzarigenin glycosides of Digitalis canariensis L. var. isabelliana (Webb) Lindinger, said mixed crystals having M.P. of about 270-276 and a toxicity of 0.45 i- 0.05 mg./kg. (cat, i.v. determined according to Hatcher-Magnus).

2. Uzarigenin mon-oside having M.P. of 241244 C., [0L] =29.2 i 2 (c=1.l2 in methanol), R =0.55 (chlo-roform/formamide) 3. Uzarigenin-D-(+)-digitoxoside having M.P. of 241- 244 C., [a] :17.2 1 2 (c=l.09, methanol), R =0.74 (chloroform/formamide) 4. A process for the preparation of cardio-active uzarigenin glycosides which comprises the steps of extracting leaves of Digitalis canariensis L. var. isabelliana (Webb) Lindinger with water and aqueous alcohol, precipitating ballast materials from the aqueous alcoholic solution, extracting the thus purified aqueous alcoholic olution in turn with ether, chloroform, and a chloroform/ alcohol mixture consisting of one part by volume of alcohol and four parts by volume of chloroform, isolating the active materials which pass over into the chloroform/ alcohol mixture, and isolating mixed crystals of uzarigenin glycosides from said active materials by dissolving said active materials in acetone, recovering a precipitate therefrom, and recrystallizing the precipitate from aqueous methanol to obtain mixed crystals of uzarigenin glycosides.

References Cited in the file of this patent UNITED STATES PATENTS 2,069,687 Stoll et al. Feb. 2, 1937 2,395,337 Marker et al Feb. 19, 1946 2,449,673 Rosen Sept. 21, 1948 2,557,916 Rosen et al June 1, 1951 2,615,884 Rosen et al. Oct. 28, 1952 

4. A PROCESS FOR THE PREPARATION OF CARDIO-ACTIVE UZARIGENIN GLYCOSIDES WHICH COMPRISES THE STEPS OF EXTRACTING LEAVES OF DIGITALIS CANARIENSIS L. VAR. ISABELLIANA (WEBB) LINDINGER WITH WATER AND AQUEOUS ALCOHOL, PRECIPITATING BALLAST MATERIALS FROM THE AQUEOUS ALCOHOLIC SOLUTION, EXTRACTING THE THUS PURIFIED AQUEOUS ALCOHOLIC SOLUTION IN TURN WITH ETHER, CHLOROFORM, AND A CHLOROFORM/ ALCOHOL MIXTURE CONSISTING OF ONE PART BY VOLUME OF ALCOHOL AND FOUR PARTS BY VOLUME OF CHLOROFORM, ISOLATING THE ACTIVE MATERIALS WHICH PASS OVER INTO THE CHLOROFORM/ ALCOHOL MIXTURE, AND ISOLATING MIXED CRYSTALS OF UZARIGENIN GLYCOSIDES FROM SAID ACTIVE MATERIALS BY DISSOLVING SAID ACTIVE MATERIALS IN ACETONE, RECOVERING A PRECIPITATE THEREFROM, AND RECRYSTALLIZING THE PRECIPITATE FROM AQUEOUS METHANOL TO OBTAIN MIXED CRYSTALS OF UZARIGENIN GLYCOSIDES. 